State of the Science: Urine, Blood and Hair Drug Testing

State of the Science: Urine, Blood and Hair Drug Testing

Toxicology: Introduction to a Cool Forensic World Carl M. Selavka, Ph.D., DABC Forensic Analytical Chemist Northeastern Bioscience Associates, LLC Charlton MA Disclaimers The opinions expressed in this presentation are those of the author and do not represent the official

positions of any agency, corporation, or scientific body. The author does not purport to provide accurate, reasonable, intelligent or timely advice. Past performance is not necessarily a guide to

future Bottom Line Up Front Diverse Investigative Power Complementary Tox = Good Alcohol Bio-Markers = ti Quality

Forensic Services it a Ce on rt ifi ca ed cr Ac

on ti The Quality Triangle Standardizati on FEPAC Forensic Science Education

Programs Quality Rectangle Educational Harmonization Certificati on Standardizati Services Accreditati

on Quality Forensic Toxicology Is Perfect Tell us About the Science of Drug Screening Fast &

Sensitive Highly Automated Drug Screening Fast & Sensitive Highly Automated Confirmation

Separate Drugs & Metabolites Incredibly Sensitive Im a Gas Chromatograph / Mass Spectrometer! I Can Detect MicroFarts

of Drugs & Metabolites! Im an even smarter Liquid Chromatograph tandem Mass Spectrometer (LC-MS/MS)! Dual Pumps Mobile Phases

Autosampler Spare Columns Sample Tray & Injector MS/MS uniquely identifies the separated drugs HPLC (Separation) and metabolites Columns in a controlled temperature zone

Toxicological Analytes of Interest Alcohol: King of the Universe (Well.At Least King of the URI Universe?) Benzodiazepines: The Pam Family of 20 Gamma-Hydroxybutyric Acid (GHB): Is it a

DFSA Bigger Problem than Roofies? Toxicological Analytes of Interest Opiates and Opioids: (Dilaudid, Percocet/dan) Fentanyl & its Analogues Cocaine: Queen for a Week Marijuana: (Old Chemotherapeutic

and New Medical Apps plus Synthetic Analogues) Sympathomimetic Amines Toxicological Analytes of Interest Hallucinogens: Stamps & Shrooms Healthy Therapeutics: Anticonvulsants &

Antiarrhythmics Happy Therapeutics: Antidepressants Toxicological Analytes of Interest All the Rest of Those Toxicologically- Important Analytes:

Carbon Monoxide Cyanide Inhalants Metals Pesticides Herbicides You Name It, Someone Was Killed Millenial Analytes of

Interest Kratom, Krocadile & Other Little Fish Designer Stimulants (Bath Salts) Legal Marijuana (26 States: Medicinal + Recreational; ALL States: CBD

(but watch out for THC) Millenial Analytes of Interest Kratom, Krocodile & Other Little Fish Designer Stimulants (Bath Salts) Legal Marijuana (26 States: Medicinal + Recreational; ALL

States = CBD) Forensic Tox Categories Compliance Testing To Maintain Privileges] Performance Testing Postmortem Examinations [ Postmortem Toxicology

Forensic Toxicology Initiation Forensic = Of, or Relating to, or Used in Legal Proceedings or Argumentation Toxicology = The Study of the Adverse Effects of Foreign Substances in Living Organisms (a.k.a. Poisons) Early 1800s: French Research Defined the Field (AND Absorptions ImportanceNow We Have ADME) Mid 1800s: The French Kept it Going with Extractions

Performance Testing Evaluation of Human Performance Can be Termed Psychomotor Performance Performance of Routine Tasks Like SPORTS! These Routine Tasks are Often Privileges Also Psychology, Toxicology & Pharmacology The Dose and History of Dosing are Critical Positive Effects vs. Negative Effects

Workplace Testing Forensic Toxicologys Workhorse Safety & Security Sensitive Positions Interface Between Privacy & Social Good Source of Forensic Best Practices The Average

Theory Absorption Man IN Distribution Metabolism Elimination OUT

Absorption Through Skin, Lung and GI Substance Enters the Blood Simple Diffusion, Active Transport Distribution Determined by Blood Flow, pKa and Lipophilicity

Leads to Different Levels In Various Tissues Metabolism Change Lipophilic Substances to Water Soluble Products Primary: Liver, Lung, Kidney, Intestine, Skin

Excretion Through Kidney (Urine) Through Liver (Bile Feces) Through the Lungs (Breath) Others - Sweat, Hair, More to Be Dangerous With Absorption and Distribution Metabolism and Excretion

Conc Plasma Urine Time Bottom Line In Middle Diverse Investigative Power Complementary Tox = Good

Alcohol Bio-Markers = Breath Tracks Blood Unusual Samples Oral Fluid Semen Sweat Body Fluid Stains Hair Finger / Toe Nails Milk Stool / Meconium

Tears Skin Oral Fluid Testing Oral Fluid Tests & U/ A Highly Complementary On-Site and Lab-Based Tests Use & Interpretive Concerns Collection Bias / Artifacts

Quality Control Issues Realities of On-Site OF PoC Tests in General: Selectivity Issues Sensitivity Issues Presumptive = Confirmation Detection Timeliness Builds

Sweat Testing Sweat Collection 3581109 Hair Testing Hair Testing Promises Indicates Multiple

Ingestions Distinguish Time Periods Difficult to Evade How big are the Windows Its All About the Half-Life (Elimination or

Oral Fluid Tracks Blood (pretty closely, anyway) Concentration (ng/mL) Absorption and Distribution Peak Quantitative Level Oral Fluid

Metabolism and Excretion Time Concentration (ng/mL) Peak Quantitative Level Oral Fluid Cutoff Level Time

Concentration (ng/mL) Qualitative Reporting Peak Quantitative Level Oral Fluid POSITIVE Cutoff Level

NEGATIVE Time Concentration (ng/mL) Window of Detection Peak Quantitative Level Oral Fluid

NEGATIVE Window of Detection POSITIVE Cutoff Level Concentration (ng/mL) Window of Detection Peak Quantitative Level

POSITIV E Oral Fluid Window of Detection NEGATIVE Cutoff Level

Its All About the Cutoff (actually) Breath Correlates to Urine Looks Farther

Drug Incorporation into Hair The Simple Model Hair Morphology Cuticle Cortex Medulla Dermal Papilla

Capillary Bed Blood Flow Undifferentiated Hair Matrix Cells U U Drawing Serum from the Capillary Bed

U Differentiation of Matrix Cells Cuticle Medulla Cortex U IRS

Detecting A Single Use ~ 0.3 mm Tip (Distal) End Root (Proximal) End Detecting Chronic Use Tip (Distal) End

Root (Proximal) End Drug Incorporation into Hair The Complex Model Complex Drug Incorporation Particles & Vapor Sweat

Sebaceous Gland Capillary Flow to the Dermal Papilla After Morrison and MacCreehan Complementary Alternatives Breath & Blood/Products = Very

Recent Use or Exposure Oral Fluid = Recent Use or Exposure Urine = Mid-Range Use or Exposure Hair = Repetitive Use or Detection Windows Hair Sweat Patch

Urine Oral Fluid Breath & Blood / Products -8 -6 -4 -2 0 2 4 6 8 Days From Last Dose

Programmatic Realities Deter Knowing & Willful Use Optimized Applications: For-Cause Incidents = Blood / Oral Fluid Changed Behaviors = Urinalysis Repetitive Use Detection = Hair Tests Legal Applicability Ask as Many Questions as Possible

to Optimize the Probative Nature of the Findings. Amphetamines DEA Schedule I or II Stimulants Synthetic Products Tablets Bottom Line In

Middle Diverse Investigative Power Complementary Tox = Good Alcohol Bio-Markers = Old & Newer Alcohol Tests [ Breath & Blood for EtOH = 6 12 hours ] Urine

for Ethanol = 8 - 24 hours o Urine for EtG days

& EtS = 2 - 4 Old & Newer Alcohol Tests [ Breath & Blood for EtOH = 6 12 hours ] Urine for

hours o Urine HO COOH O HO O HO

days EtG C H for 2 5 O O

O Ethanol = 8 - 24 EtG & EtS = 2 - 4 FAEE Ethyl

Ethyl myristate C2H5 C2H5 Ethyl palmitate Alcohol Detection Windows Hair Fatty Acid Ethyl Esters (FAEEs) Hair Ethyl Glucuronide (EtG) Urine

EtG & EtS S-B PEth Serum-Blood CDT Serum-Blood AST/ALT

Serum-Blood 5- HTOL Month 6 Month 5 Month 4 Month 3 Month 2

Month 1 Days 1-7 Windows of SC Detection (WoDs in Days / Weeks / Months) Week 2 Forensic Hair Test Extensions Hair alcohol biomarkers (FAEEs & EtG):

unique & powerful alternative investigative Categories of Alcohol Consumption Classification Teetotaller according to amount of drinking (not unequivocal; bio-diversity, plus depends on gender): Social drinkerStrict abstinence

Non-drinker } 0 g/day < 1 g/day Moderate alcohol consumption Alcohol Abuse Medium social consumption Alcohol Addiction }

1-20 g/day 21-60 g/day (1) (2) Risky alcohol consumption 61-120 g/day Diagnostic tasks alcohol consumption >120 g/day Excessive ) Measure maintenance of abstinence or control for problem drinkers

) Discriminate between social drinking & alcohol abuse Note: 60g EtOH = 7 - 8 beers / shots per day! Units/Grams Equivalent to Drinks Units Grams EtOH Type/Amount of Drinks 1 8

1 drink (shot, 12oz. beer, glass of wine) 9.75 78 750 ml bottle of wine with 13% EtOH 19 152

475 ml (pint) of 80 proof (40%) Gin/Vodka 23.5 190 475 ml (pint) of 100 proof (50%) Whiskey 30 240 750 ml bottle of 80 proof (40%) Gin/Vodka

37.5 300 750 ml bottle of 100 proof (50%) Whiskey HO O Alcohol consumption bio-markers in hair

HO Fatty acid ethyl esters (FAEEs) Group of more than 20 substances 4 esters selected Ethyl myristate Ethyl palmitate Concentration sum CFAEE used for diagnosis CFAEE range 0.1 145 ng/mg

depending on EtOH dose Ethyl stearate Ethyl oleate Et C2H5 O O O O

O O O O C2H5 FA Eth C2H5

Et C2H5 Et C2H5 E Ethyl glucuronide (EtG) State of knowledge and practical use

CEtG range 1 8000 pg/mg depending on EtOH dose HO HO HO Biochemical basics widely explored Direct alcohol bio-markers COOH Discriminate between social and excessive drinking O EtG

Validated methods for analytical determinations O C2H 5 Established practical applications; still in test phase Experiences with reliability and sources of errors O FAEE O C2H5 Ethyl myristate

Incorporation of FAEEs in hair Sebum Incorporation is mainly from sebum Detection in skin surface lipids by sweat patches, wipe test and Sebutapes up to four weeks after abstinence begins (Pragst et al. 2004, Nadulski et al. 2007)

High concentrations in n-heptane washings of hair Delayed incorporation from surrounding fat tissues Accumulation in the hair shaft outside of the root, typical concentration profile of segments Dominating route Conc. in hair) (ng/mg Formation from ethanol in the sebum glands

and incorporation in hair from sebum Formation of FAEE in the hair root Blood vessels Incorporation of FAEE from blood Distance from hair root (cm)

Incorporation of sweat EtGEtG ininhair Mechanism still being researched: (a) Incorporation from blood - low incorporation rate because of anionic and hydrophilic structure - Not totally improbable, since concentrations in hair are very low

(b) Incorporation via sweat - Preferred mechanism - Excretion of EtG in sweat: Schummer et al., Ther. Drug Monit 2008 Low excretion rate (acidic compound) Concentrations two orders below blood (1.7-35 ng/mL) EtG in blood Complications Variations in sweating depending on

temperature, physical exercise & individual bio-diversity On monthly basis: 60 120 - Low AUCEtOH for moderate and social drinking - Strong increase of AUCEtOH above 60 g per day - Essential influence of drinking habits

Excellent basis for high discrimination power of the hair test for alcohol abuse Scheme for combined interpretation of FAEE and EtG Original 2009 cut-offs: FAEEs 0.5 ng/mg, EtG 25 pg/mg. SoHT 2011 criteria: - Levels for Abstinence = FAEE 0.20 ng/mg, EtG not detected

(<2.4-pg/mg) to 7-pg/mg - Increased cut-off values (1.00 ng/mg and 50 pg/mg for SIX & THREE MONTHS!) Pre-Analytical Fun Factors Sample Provenance Who, Why, When Who = Generally Adversarial Why = Custody, Visitation, Defendant When = Unannounced is Best

Integrity SC+Self-Report, Production SC+Self-Report = Competent Confounding Forensic Factors Lab Based: Availability of ID Standards Bath Salts & Cannabinomimetics Phosphatidyl Ethanol (PEth)

Congeners Deuterated Analogues Interpretive Need: Comparable Result DBs Hair Treatment Categories Dye = Permanent (grows out) or Temporary (washes

out) Colour = IBID. (but usually Temporary) Bleach = Peroxide-based (includes highlights and brightening) Bottom Line In

Middle Diverse Investigative Power Complementary Tox = Good Alcohol Bio-Markers = FutureForensic Realities Point of Collection (PoC) Testing Point of Care Equivalency (Lab)

Investigative BaRD (PC) < PoE < RDSC < BAC from BrAC DUID from Breath EtG and EtS in Oral Fluids

Another Confounding Factor Availability of Identification Standards Bath Salts & Cannabinomimetics Phosphatidyl Ethanol (PEth) Congeners Deuterated Analogues Comparable Result Databases Lack of Abusive Clinical

Forensic Interface Factors Gauging Precision Evidentiary Limits Must Avoid Specimen Consumption Sample Heterogeneity (e.g. Hair, Stains) Peer Review Multiple Deficiencies

Evidence Collection training, experience Bottom Line Diverse Investigative Power Complementary Tox = Good Alcohol Bio-Markers = Acknowledgments

- URI Forensic Science Partnership - Tripler FTDTL, NMS, NYSDCJS, MSPCLS, NERFI, USACIL, AFDTL, Drinkers, ADL EU/UK

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